Examination of live samples provides access to the dynamics of cellular and sub-cellular processes. The study of small-scale cellular processes has typically been carried out using light microscopy.
In some cases, the complexity of the processes is such that tools are required to isolate specific types of processes which can be studied in isolation, or in combination with a small number of other processes. The use of labels (in the form of fluorescent molecules in fluorescence microscopy) has provided tools of this nature.
A label may be extrinsic (that is, added to a sample) or intrinsic (such as a molecule which is present in the sample that is inherently distinctively coloured or fluorescent).
A fluorescent molecule has a specific excitation spectrum, being more strongly excited at some wavelengths and less strongly excited at others. It also has a specific emission spectrum, emitting more intensely at some wavelengths, and less intensely at others. The excitation and emission spectra may range from the ultraviolet to the infrared.
A wide range of fluorescent labels have been developed from chemical molecules, such as Rhodamine and Fluoroscein. Further fluorescent labels have been developed from molecules found in luminescent organisms, for example the Aequorea Jellyfish which has provided the Green Fluorescent Protein (GFP), and various corals providing DsRed and HcRed. These have been termed AFPs (Aequorea Fluorescent Proteins).
The fluorescent labels may be associated with specific molecules of interest (DNA, RNA, proteins, carbohydrates, antibodies, etc). Alternatively they may be made to be sensitive to certain characteristics (such as ionic concentration, pH, voltage potential, temperature, the presence of a specific enzyme, the presence of specific enzyme substrates, force), altering their fluorescent properties according to these characteristics. These labels may be introduced into cells by passing through the cell membrane or by injection. Alternatively they may be formed internally as part of the normal functioning of the cell, in the case of the genetically encoded labels such as the AFPs.
In a known apparatus for imaging samples as shown in FIG. 1a, a microscope 10 is fitted with a light source 12 for illuminating a sample 4b. It may be in the form of a lamp, laser or light emitting diode, for example. The microscope is fitted with optical filters 14 so that the light emanating from the sample may be observed at selected wavebands. Examination of the spatial and temporal distribution of light from the sample may provide information on the structure and dynamics of the sample.
Phase contrast illumination may be employed to enhance imaging of thin samples. Polarised light may be used to permit the visualisation of very thin samples with small refractive index changes, for example using differential interference contrast (DIC) techniques.
The microscope is fitted with an image acquisition system, comprising a light sensitive detector 16 (sensitive from the ultraviolet to the infrared) such as a CCD camera, and recording means such as a video recorder or computer 18 with a memory device 20, so that dynamic behaviour of the sample may be captured and analysed offline. For example, the velocity, distance traveled and path of moving parts of the sample may be monitored.
The system may employ a focus drive mechanism for altering the position of the imaging focus plane. Various algorithms may be used to establish and maintain the optical focal plane. Volumetric (XYZ) and volumetric time series (XYZT) data may be acquired using deconvolution techniques. By selecting suitable excitation wavebands and/or selecting suitable optical filter sets, volumetric multi-wavelength (XYWZ) and volumetric multi-wavelength time series (XYWZT) data may be acquired.
Operations of the microscope are controlled by a microscope controller 24 in response to commands from processing means in the form of computer 18. Instructions may be entered into the computer by a user using keyboard 26, a mouse 28 and a display 30.
By way of example, FIG. 1a shows control lines extending from the controller to the microscope for aspects such as filter selection along line 32, selection of an objective lens 34, 34′ along line 36, focus along line 22, a liquid dispenser 38 along line 40, an environmental unit 42 along line 44 for modifying environmental conditions of a sample 46, and a sample transport unit 48 for moving the sample 46 relative to a lens of the microscope by control via line 50.
The apparatus may also include an activation light source 52 for generating an activating light beam 54 for use as discussed below. The direction of the activating light beam may be adjusted by a guide 56.
The activating light beam 54 is incident on a dichroic mirror 60. The mirror directs the beam towards an objective lens 34, and the beam is then incident on the sample 46. Light emanating from the sample 46 passes back through the objective lens 34, but is not diverted by mirror 60 so that is passes through a filter 14 before impinging on detector 16. An output signal from the detector 16 is fed to the computer 18 along line 62.
The environmental unit 42 may control the temperature of the sample, and/or the composition and flow of gas over the sample, for example.
Software loaded onto the computer enables it to acquire image data carried on the output signal from the detector.
The software also allows the user to select parameters for the operation of various aspects of the apparatus, such as the activating light beam, for example.
The controller allows the activating light beam to be directed by the setting of one or more of the following parameters:                the one or more regions of interest on the sample (location, size and shape)        the wavebands        the power level        the time of start of activation        the duration of activation        the number of repeat activation cycles        the delay between activation cycles.        
The computer permits recording of the image data from the detector via a digitiser at a given data rate. The filter sets in the microscope and the waveband of the illuminating light source 12 may be controlled to permit acquisition of data sets of one or more wavelengths.
A known apparatus for imaging biological samples including fluorescent labels is shown in FIG. 1b. It is similar to the apparatus shown in FIG. 1a. However, instead of illumination light source 12, it includes an excitation light source 11, which generates a light beam 55. This light source is capable of exciting one or more fluorescent labels present in the sample by irradiating them with one or more specific wavebands. Filters 14 may be chosen so as to pass light emitted by labels at selected wavebands.
FIG. 1c illustrates attachment of a fluorescent label 2, 2′ to a component of interest 4, 4′ in a sample 6. Multiple labels may be used to provide information on the coincident localisation of labelled components, revealing, for example, the organization of the cytoskeleton of a cell.
The excitation light source 11 employed in the apparatus of FIG. 1 may be pulsed and the detector may be fitted with a gating device, so that the time taken for a fluorescent label to emit light following the pulse may be used to distinguish between different labels in a technique termed fluorescent lifetime imaging (FLIM) [ref. R. Cubedda, J. Phys. D: Appl. Phys., 2002, Vol. 35, R61-R76].
An activating light beam 54 may enable a deeper understanding of the dynamic processes in a sample such as a living cell (in many cases this may be based on the excitation light source) to be obtained. The activating light beam may be directed to portions of the sample in such a manner that the intense light from this beam alters the sample and/or bleaches labels present in the sample and reduces their fluorescence. By observing the subsequent changes in the light emanating from this region, and/or elsewhere in the sample, information can be obtained on mechanisms of interaction and/or exchange of various labelled components.
The beam may act to perforate a cell wall to allow the entry of an external agent, to dissect all or part of the cell, to destroy all or part of the cell, or to change the environment of the sample.
A graph plotting the intensity of fluorescence at a bleached region against time is shown in FIG. 1d. 
For example, the rapid recovery of fluorescence at the bleached portion suggests to what extent the label is free to return to the region. The mechanism, diffusion or local synthesis, may be determined by estimation of diffusion rates and the mobile fraction [ref. AxelRod, Biophys. J., 1977, Vol. 18, pp. 129-131.].
Further analysis and experimentation may provide access to information about molecular binding rates, assembly/disassembly and transport mechanisms [ref. J. Lippincott-Schwartz et al, Nature Supp Imaging in Cell Biol, September 2003, S7-S14.]. Such techniques are popular to explore the dynamics and regulation of processes in cells, for example protein trafficking, lifetime and fate (recycling and breakdown).
Conventionally, such bleaching techniques may be applied as a “bleaching protocol” comprising four phases as illustrated in FIG. 1e:                 (1) a pre-bleach phase in which the sample is imaged using normal fluorescence for a period of time;        (2) a bleach phase during which the activating light beam is directed to illuminate a predefined region with light of a specific wavelength with a specified higher power level for a certain period of time;        (3) a post-bleach phase in which the sample is again imaged using normal fluorescence for a period of time; and        (4) an analysis phase in which the data which has been collected is processed to determine parameters of interest, such as diffusion rate and mobile fraction according to fluorescence intensity in various regions on the image at various time points.        
A set of related techniques are commonly referred to as FRAP (Fluorescence recovery after photo-bleaching). Variants of the basic protocol may be used to further explore the system, by repeated bleaching of the same region, and bleaching other portions etc (FLAP, FLIP, iFRAP) [ref. J. Lippincott-Schwartz et al, see above; Phair et al, Nature, 2000, Vol. 404, pp. 604-609.].
Alternatively, a label in the sample, the sample itself or its environment may comprise a caged compound that releases an active group when illuminated by the activating light beam—a process known as uncaging [ref. J. C. Politz, Trend Cell Biol., 1999, Vol. 9, pp. 284-287.]. The active group may be a fluorescent label. Alternatively, the active group may not be a fluorescent label, for example the active group may instead affect the pH of the sample. The active group may have an indirectly visible or otherwise measurable effect on the sample.
In addition a technique known as pattern photo-bleaching may be using to observe changes in structure [ref. J. Ellenberg et al, Nature Supp Imaging in Cell Biol, September 2003, S14-s19.]. In this technique, the bleaching process is used to introduce visually distinctive landmark patterns on part of the sample, for example a grid, movement of which may be tracked over time to understand, for example, mechanisms of membrane deformation or shear.
Recent developments in fluorescent probes has resulted in new labels (photo-switchable labels) which are sensitive to the incident light, in such as way that they change their optical properties, altering their emission spectrum and/or their excitation spectrum. Examples include the photo-activatable GFP (PA-GFP) and Kaede [ref. J. Lippincott-Schwartz et al, see above.]. Furthermore, some of these probes allow their properties to be altered reversibly or irreversibly. Such probes may be activated (made to emit more intensely), or quenched (made to emit less intensely) in different wavebands. For example the kindling FP KFP1 [ref. D. M. Chudakov et al, J. Biol. Chem., 2003, Vol. 278(9), pp. 7215-7219.].
Instrumentation has been developed with an adapted activating light beam to excite photo-switchable labels and force them to undergo the change in optical properties. This may, for example, be useful in revealing or hiding labelled components during the course of an experiment and in studying long term processes such as organism development [ref. D. M. Chudakov et al, Nature Biotechnology, February 2003, Vol. 21, pp. 191-194.].
Hereinafter, the term photo-modification will be used to include photo-bleaching and its variants (FRAP, iFRAP, FLIP, FLAP), photo-activation, photo-switching, and pattern photo-bleaching.
Conventional control of an activating light beam involves setting up a number of activation parameters and requires the user to be able to estimate the appropriate values. In general, these are determined by trial and error using surplus material. It may be desired to set the depth of activation to be say 30% (as is the case in the illustration of FIG. 1d) and the appropriate laser power setting and duration must be determined to achieve this. Similarly, the recovery time of the curve will determine the appropriate sampling regime in the post-bleach period (number of images, interval between images) which in turn is determined by the diffusion coefficient.
These activation parameters must be defined prior to the experiment. They cannot be adapted to suit changing conditions. This means that the approach can only be applied to changing samples by trial and error. Thus fast changes and/or rare events can be extremely difficult to study.
In addition, in existing photo-bleaching procedures, the recovery after bleaching prevents long time course experiments (10 mins+) in the tracking of change since the labels recover over shorter periods and thereafter there is no discernible bleached area.